The present study used the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo/) to retrieve expression profile datasets. The search term used was: 'Ventilator lung'.

In total, 280 male ICR mice (aged 8-10 weeks, weighting 25-30 g) were purchased from the Animal Experimentation Center of the Second Military Medical University. All mice had free access to water and food and were housed at room temperature (20-22°C) with 30-70% humidity under a 12-h light/dark cycle. All experimental protocols were approved by The Shanghai Jiaotong University School of Medicine and the methods were conducted in accordance with the institutional guidelines (ethical approval nos. XHEC-C-2017-058 and XHEC-F-NSFC-2018-057).

Mice underwent ventilation and were intraperitoneally administered with 1 mg/kg LPS (LPS + ventilation group) or saline (ventilation group) (2). After 12 h, the mice were anesthetized with ketamine (70 mg/kg) and xylazine (10 mg/kg) (19), and connected to a ventilator (Inspira, Harvard Apparatus Ltd.) following tracheotomy (10 ml/kg at 150 breaths/min) for 4 h. SPHK1 inhibitor SKI II (50 mg/kg) or ROCK1 inhibitor RKI-1447 (10 mg/kg) were injected intraperitoneally 1 h prior to ventilation. All animals were sacrificed following ventilation, and bronchoalveolar lavage (BAL) fluid or lung tissues were collected. The blood samples were centrifuged at 956 × g for 10 min at 4°C. Lung samples were fixed in 4% paraformaldehyde for 1 day at room temperature, employed to determine the lung wet-to-dry (W/D) ratio or stored at −80°C until use.

For the first set of experiments, the present study aimed to investigate the effects of SPHK1 inhibition on VALI. The mice were randomly assigned to five groups (n=7 per group): i) Saline administration followed by spontaneous breathing (non-ventilated group); ii) LPS administration followed by spontaneous breathing (non-ventilated + LPS group); iii) saline followed by ventilation (ventilated group); iv) LPS administration followed by ventilation (ventilated + LPS group); and v) LPS and subsequent SPHK1 inhibitor administration with ventilation (ventilated + LPS + SPHK1 inhibitor group).

For the second set of experiments, the present study aimed to investigate the effects of ROCK1 inhibition on VALI. The mice were randomly allocated to the following five groups: i) Saline administration followed by spontaneous breathing (non-ventilated group); ii) LPS administration followed by spontaneous breathing (non-ventilated + LPS group); iii) saline administration followed by ventilation (ventilated group); iv) LPS administration followed by ventilation (ventilated + LPS group); and v) LPS and subsequent ROCK1 inhibitor administration with ventilation (ventilated + LPS + ROCK1 inhibitor group).

BAL fluid collection was performed three times with 1 ml of saline as previously described (12). The total cell counts were determined as previously described (20) and the protein concentration of BAL fluid was analyzed using a commercially available bicinchoninic protein assay kit.

The index of pulmonary edema formation was calculated by obtaining the lung W/D weight ratio. The lung weights prior to and following drying were used to calculate the lung W/D ratio as previously described (13).

Evans blue dye was injected intravenously at 30 mg/kg 1 h prior to the termination of ventilation to assess vascular leak, according to a previously described protocol (13).

The left lung tissues were fixed in 4% paraformaldehyde at room temperature for 1 day, and then stained with hematoxylin for 5-10 min at room temperature and eosin (Beyotime Institute of Biotechnology) for 1-2 min at room temperature. The pathogenesis of lung samples was graded by two pathologists in a blinded manner as previously reported: i) 0=Normal tissue; ii) 1=minor inflammatory alterations; iii) 2=mild to moderate inflammatory alterations without marked damage in the lung architecture; iv) 3=moderate inflammatory injury with thickening of the alveolar septa; v) 4=moderate to severe inflammatory injury with the formation of nodules or areas of pneumonitis; and vi) 5=severe inflammatory injury with total obliteration of the field. The mean score was reported per section.

The content of S1P in the serum was determined via ELISA using a commercial kit, according to the manufacturer's instructions (cat. no. abx585002; Abbexa Ltd.).

The activities of ROCK1 in the lung tissue and in mouse lung vascular endothelial cells (MLVECs) were analyzed via ELISA using a commercial kit, according to the manufacturer's instructions (cat. no. STA-416; Cell Biolabs, Inc.).

MLVECs isolation and culture were performed using a modified method as previously described (9). Briefly, the anesthetized mice were perfused with DMEM (Gibco; Thermo Fisher Scientific, Inc.) from the right ventricle to remove blood from the lungs. Then, the subpleural pulmonary tissue was cut into pieces and cultured in DMEM with 20% FBS under 5% CO₂ at 37°C for 60 h. The tissue was removed and the adherent cells at passages three and four were used. For cyclic stretch, MLVECs (1×10⁶ cells/well) were seeded onto collagen I-coated Bioflex^® 6-well culture plates (Flexcell International Corporation) and grown to 80% confluence. MLVECs were then exposed to cyclic stretch (8% linear elongation, sinusoidal wave, 30 cycles/min) for 4 h, as previously described (21), by using the Flexcell^® FX-5000 Tension system (FX5K; Flexcell International Corporation). The cells that did not receive cyclic stretch were placed in the same incubator for 4 h at 37°C.

Endothelial cell permeability was analyzed according to a previously published protocol (22). The assay was based on the high affinity binding of an avidin-conjugated FITC-labeled tracer to biotinylated extracellular matrix proteins immobilized on the bottom of culture dishes covered with an MLVEC monolayer. The BioFlex plates (Flexcell International Corporation) were coated with biotinylated gelatin (Sigma-Aldrich; Merck KGaA), and MLVECs were seeded in the wells (1×10⁶ cells/well). After cyclic stretch, cells were fixed with 3.7% formaldehyde at room temperature for 10 min, and FITC-avidin (25 µg/ml) was added to the culture medium for 3 min at room temperature. After washing, elastic bottoms of plates were excised with a scalpel and transferred onto a microslide. The fluorescence was then analyzed under a microscope (magnification, ×20; Olympus Corporation).

For western blotting, the lung tissues and MLVECs were homogenized in cold RIPA lysis buffer (Beyotime Institute of Biotechnology) containing proteinase and phosphatase inhibitor cocktail (Roche Diagnostics). Equal amounts of protein extract (30-40 µg) were separated by 8-12% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). After blocking in 5% non-fat dry milk for 2 h at room temperature, the membranes were probed overnight at 4°C with anti-SPHK1 (cat. no. ab71700; 1:500; Abcam), anti-phosphorylated (p)-myosin phosphatase target subunit 1 (p-MYPT1; cat. no. 5163S; 1:1,000; Cell Signaling Technology, Inc.), anti-MYPT1 (cat. no. 2634S; 1:1,000; Cell Signaling Technology, Inc.), anti-RhoA (cat. no. ab187027; 1:3,000; Abcam) and anti-β-actin (cat. no. sc-47778; 1:500; Santa Cruz Biotechnology, Inc.) antibodies. After washing, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies (cat. nos. ab6721 and ab6728; 1:5,000; Abcam). The antibody-reactive bands were visualized via an enhanced chemiluminescence western blotting detection system (EMD Millipore). The membranes were visualized using the UVP Bio-Imaging system. Densitometric analysis was performed using Quantity One software (version 4.6; Bio-Rad Laboratories, Inc.).

RhoA activity was measured via a pull-down assay using glutathione S-transferase (GST) fusion of Rho-binding domain (RBD) (Cell Signaling Technology, Inc.) according to the manufacturer's instructions. Following treatment, lung tissues and MLVECs were collected and lysed in cold RIPA lysis buffer. Supernatants were incubated for 2 h at 4°C with GST-RBD coupled beads. Centrifuge the samples at 3,824 × g for 10-30 sec and precipitates were washed three times with RIPA lysis buffer and suspended in 2X SDS sample buffer (Cell Signaling Technology, Inc.). The activated RhoA bound to the beads or total RhoA in cell extracts was detected using western blot analysis with a RhoA antibody following the aforementioned protocol.

Total RNA isolated from mouse lungs tissue using TRIzol^® reagent (Thermo Fisher Scientific, Inc.) was used for RT. Subsequently, qPCR was performed using SYBR^® Premix Ex Taq (2X; Takara Bio, Inc.) with a StepOne Plus system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The amplification conditions were as follows: Initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec, 60°C for 30 sec and 72°C for 30 sec, with a final extension at 72°C for 5 min. Mouse β-actin served as an internal control, using the 2^−ΔΔCq method (23). The sequences of the primers used are listed in Table I.

Data are presented as the mean ± SEM. Normal distribution was assessed by Shapiro-Wilk test. Statistical significance was measured according to sample distribution and homogeneity of variance. Statistical comparison between two groups were determined by Student's t-test for normally distributed data and by Mann-Whitney U test for non-normally distributed data. One-way ANOVA with Bonferroni's post hoc test was performed to compare multiple groups using SPSS 19 (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

To determine the expression levels of SPHK in ventilated mouse lung tissues, the present study analyzed the gene expression profiles of the GEO mouse VALI datasets GSE58169 (24), GSE29920 and GSE9208 (25). Collectively, the analysis of these data revealed that the mRNA expression of SPHK1, rather than SPHK2, was significantly upregulated in ventilated mouse lung tissues (Fig. 1A and B). To verify these findings, the expression of SPHK1 was investigated by RT-qPCR and western blotting. As shown in Fig. 1C and D, there were no significant differences in mRNA and protein concentration of SPHK1 in LPS alone or ventilated alone group. However, in the ventilated + LPS group, the expression of SPHK1 was significantly increased compared with that in the non-ventilated group. SPHK1 inhibitor had no effects on the mRNA and protein expression of SPHK1. Furthermore, consistent with the increase in SPHK1 expression, the levels of pulmonary S1P were significantly increased in the ventilated + LPS group, but decreased following treatment with SPHK1 inhibitor (Fig. 1E).

In the present study, it was investigated whether SPHK1 inhibitor could exhibit a protective effect on the two-hit of model of VALI. As shown in Fig. 2A and B, LPS or ventilation alone induced minor non-significant lung injury compared with the non-ventilated group. In line with our previous study (13), low tidal volume ventilation did not exert significant lung injury. However, when combined with LPS, ventilation significantly induced lung injury, indicating a synergistic effect of these two stimuli. In addition, the degree of lung injury in the two-hit VALI models was significantly attenuated following treatment with SPHK1 inhibitor. Similar results were observed in the lung W/D weight ratio and the Evans blue assay (Fig. 2C and D). Ventilation + LPS led to increased lung W/D ratio and Evans blue leakage from the vascular space into the lung parenchyma, which were significantly alleviated following treatment with SPHK1 inhibitor.

H&E staining was then performed to evaluate the histology of tissues (Fig. 3). Histopathological analysis revealed that mice in the two-hit VALI group (ventilated + LPS) exhibited diffuse interstitial edema and infiltration around the pulmonary vessels; alveolar and interstitial edema were also observed compared with the LPS and ventilated groups. Quantitative analysis demonstrated a significant increase in the lung injury score in the ventilated + LPS group. Following treatment with SPHK1 inhibitor, lung injury induced by ventilation + LPS was significantly attenuated.

RhoA and its target protein, ROCK, are involved in the calcium-sensitizing signaling pathway associated with the cytoskeletal contractile response via the activity of myosin ATPase (26). ROCK mediates the phosphorylation of myosin light chain, ultimately resulting in the reorganization of actin myosin, tension fiber formation and cell contraction (27). MYPT-1 is the regulatory subunit of myosin light chain phosphatase, which functions as a downstream target of ROCK (28). The present study investigated the activation of RhoA and ROCK1, and the phosphorylation of MYPT-1 in the two-hit model of VALI. As presented in Fig. 4, LPS or ventilation alone had no significant effect on RhoA and ROCK1 activity, and on the phosphorylation levels of MYPT1. By contrast, mice in the two-hit VALI group (ventilated + LPS) exhibited an increase in RhoA and ROCK1 activity, and MYPT1 phosphorylation compared with the non-ventilated + LPS or ventilated groups. Additionally, SPHK1 inhibitor treatment significantly attenuated the increases in RhoA and ROCK1 activity, and the phosphorylation levels of MYPT1 in the lung tissues from mice in the ventilated + LPS group. The present results suggested that SPHK1 inhibitor suppressed the activity of the RhoA/ROCK signaling pathway in animals with VALI.

As lung endothelial cell hyperpermeability is caused by endothelial overdistension during ventilation (1,29), the effects of cyclic stretch and LPS were examined, and the effect of SPHK1 inhibition on the activation of the RhoA/ROCK pathway was investigated in primary cultured MLVECs. In the present study, MLVECs were subjected to LPS treatment for 12 h and then exposed to cyclic stretch (8% elongation) for 4 h. Consistent with the findings in vivo, LPS or stretch alone led to minor changes in the activation of RhoA and ROCK1, and the phosphorylation of MYPT1. On the contrary, MLVECs subjected to the combination of stretch and LPS treatment exhibited significant increases in the activity of RhoA and ROCK1, and in the phosphorylation levels of MYPT1. Treatment with SPHK1 inhibitor significantly inhibited the activation of RhoA and ROCK1, and the phosphorylation of MYPT1 induced by cyclic stretch + LPS (Fig. 5).

The protective effects of SPHK1 inhibitor against stretch-associated endothelial hyperpermeability were further assessed by a permeability assay. As presented in Fig. 6, LPS or mechanical stretch alone had limited effects on endothelial permeability. By contrast, analysis of the permeability sites in the LPS + stretch-challenged endothelial monolayers revealed the presence of FITC-labeled avidin, which entered cells via weakened cell junctions and stretch-induced paracellular gaps; the combination of stretch and LPS treatment markedly increased endothelial monolayer permeability to FITC-labeled avidin. In addition, SPHK1 inhibitor treatment markedly decreased the cellular entry of the fluorescent probe via intercellular junctions, indicating that inhibition of SPHK1 attenuated the disruptive effects of LPS + cyclic stretch on the permeability function of endothelial cells in vitro.

To assess the role of the RhoA/ROCK pathway in the two-hit model of VALI, mice were treated with ROCK1 inhibitor RKI-1447 (10 mg/kg) prior to ventilation. As presented in Fig. 7A and B, ROCK1 inhibitor exerted a significant protective effect on the two-hit model of VALI as assessed by measuring the cell count and the protein levels in the BAL fluid. The results of the lung W/D ratio and Evans blue index analyses also supported these findings (Fig. 7C and D). In addition, H&E staining was performed to evaluate the effects of ROCK1 inhibitor on lung histology (Fig. 8). The degree of lung injury induced by LPS and ventilation was significantly attenuated by ROCK1 inhibitor treatment.

Additionally, the present study investigated the effects of ROCK1 inhibitor on the ROCK1/MYPT1 pathway in vivo. As presented in Fig. S1, ROCK activity was suppressed in response to ROCK1 inhibitor, and the levels of p-MYPT1 were also downregulated.

The present study investigated the effects of ROCK1 inhibitor on endothelial permeability in vitro. As presented in Fig. 9, ROCK1 inhibition by RKI-1447 (1 µM) could inhibit the entry of the fluorescent probe in cells in the stretch + LPS group. The present results suggested that inhibition of ROCK1 attenuated the barrier-disruptive effects of LPS treatment and mechanical stretch in vitro. In addition, in MLVECs, the activity of ROCK1 and the phosphorylation levels of MYPT1 were reversed by treatment with ROCK1 inhibitor (Fig. S2).